Plant Mutation Reports Vol. 1, No. 2, December 2006 | IAEA

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J. Neil Rutger in his “laboratory” in September 2002, at Stuttgart, Arkansas, observing an early flowering indica mutant line (left) and its late flowering parent (right) (see page 9)

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First of all, I would like to inform you that the Plant Mutation Reports (PMR) is now indexed in the CAB ABSTRACTS and GLOBAL HEALTH databases, run by the CABI, Wallingford, UK. This is not only an important recognition of the publication and, ultimately, the quality of your papers, but also provides a great opportunity for the broad dissemination of papers published in PMR.

Secondly, it is a great honour to include a review paper from Dr. J. Neil Rutger, former director of Dale Bumpers National Rice Research Center USDA-ARS-SPA, USA. Dr. Rutger’s paper summarizes the extraordinary success of his 30 years of work on induced mutations in rice genetics and breeding, together with his contribution to the Joint FAO/IAEA Programme. In particular, you will learn how a single induced mutation can contribute to the substantial yield increase in rice. We are aware that great success has also been achieved in rice as well as in other crops by various groups around the world; we invite you to submit papers of this kind to high- light your accomplishments or summarize your professional careers in mutation induction, application or basic studies on mutagenesis.

Thirdly, I would like to share with you our plan for further improvement of the qual- http://www-naweb.iaea.org/nafa/index.html ISSN 1011-260X

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low); (2) We are planning to transform the cover appear- ance and paper format into the style of a scientific jour- nal; (3) For expanding the manuscript’s source and quality, we decided that all final technical papers from the Agency’s coordinated research projects (CRPs) and regional and interregional technical cooperation projects (TCPs) in the field of plant breeding and genetics be published in the PMR. (4) We will also strive for a broader distribution of the PMR; free electronic subscription will be granted to all interested institutions and individuals.

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Plant Mutation Reports Editorial Board

We are planning to establish an editorial board for the Plant Mutation Reports. It will consist of about 15 editors / associate editors specialized in the following fields of plant research: (1) DNA damage, repair and mutagenesis; (2) Insertion mutagenesis; (3) Experimental mutagenesis with artificial mutagens; (3) Crop breeding and genetics with induced mutations; (4) Ge- nomics and molecular genetics of induced mutations; (5) Mutational analysis and mutant germplasm.

Interested scientists, please submit a short C.V. (2-3 pages) to [email protected] for consideration; final selection will be made on the basis of both the candidate’s expertise and balance of each field.

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The author’s 30 years of achievements with induced mutations in rice genetics and breeding are summarized, beginning with temperate japonica mutants in California, and then continuing to tropical japonica and indica mutants in Arkansas. Through- out these studies, emphasis has been on selecting agronomically useful mutants such as semidwarfism and early maturity.

The author notes that to realize the full value of mutants, induction should be closely followed by evaluation of the mutants and then their integration into cross-breeding programs.

Evaluation and integration should be done concurrently insofar as possible. The best example was induction and release of Calrose 76, the first semidwarf table rice cultivar in the USA.

Subsequent evaluation and integration steps by rice breeders have resulted in 25 improved semidwarf cultivars that trace their ancestral source of semidwarfism to Calrose 76: 13 in California, 10 in Australia and 2 in Egypt. Since 1993 induced mutation has been used to develop and release numerous tropical japonica mutants at the Dale Bumpers National Rice Re- search Center in Stuttgart, Arkansas. In the last six years induced mutation has been used to develop indica rice germplasm adapted to the USA. Previous and current induced mutants were used to establish the Genetic Stocks-Oryza Collec- tion (GSOR) in 2003. Over 50 rice mutants collected by the author and his associates have been released as germplasm or entered into GSOR. The author has had extensive involvement with IAEA in participation in international symposia, Research Coordination Meetings, and as a mutation breeding consultant in IAEA activities in Latin America and Asia.

Key words: Rice mutants, temperate japonica, tropical japonica, indica, cross-breeding, genetic stocks, improved germplasm

Introduction

My 30 years of experience with rice mutants started in 1971 in Davis, California so it might seem like that I cannot subtract very well and should say 35 years, but in 1989 I began an administrative assignment which took me away from rice research for nearly 5 years. I was fortunate to be able to return to full-time rice research in late 1993 at the Dale Bumpers National Rice Research Center (DB NRRC) in Stuttgart, Arkansas, with most of my emphasis again on rice mutants. Throughout my career I have concentrated on selecting agronomically useful mutants such as semidwarfism and early flowering, with occasional detours into mutants such as male steriles and marker genes as genetic tools. My “laboratory” for induced mutation studies has been the rice field, since that is where useful mutants eventually will have to pass mus- ter. In the last decade I have ventured into a base- broadening program with indica rice that is, developing indica rice for adaptation to our japonica rice-growing nation. Once again, in the indicas, induced mutation is playing a useful role.

During most of my 30 years I literally walked over or ignored a lot of what I called “curio” mutants, such as extreme dwarfs which were only 20 cm tall-a real prob- lem when the irrigation water is 25 cm deep!-albino mutants, etc. In the last 5 years I have come to realize that these “curio” mutants can have value as genetic stocks for basic research studies, so my colleagues and I began collecting them for our Genetic Stocks-Oryza (GSOR) Collection which was founded at Stuttgart in 2003.

California temperate japonicas

The author’s introduction to useful applications of induced mutation in rice genetics and breeding was in the early 1970s in California, with the development and release of the first semidwarf table rice cultivar in the USA, Calrose 76 (Rutger et al., 1977). Prior to the author’s arrival in 1970, Dr. Chao-hwa Hu of Taiwan, who had experience with induced mutation in his country, had received an IAEA research award to study in California. In advance of his arrival, Dr. Hu asked Dr. C.O. Qualset of the University of California Davis (UCD) to mutagneize seeds of leading California rice cultivars. Dr. Qualset ar- ranged for the seeds to be mutagenized at UCD and planted by Dr. W.F. Lehman at the UC Imperial Valley Field Station in southern California. When Dr. Hu’s Cali- fornia visit was delayed the M1 seeds were stored at the Imperial Valley Field Station until the author picked them up and planted them at UCD in 1971. The semidwarf plant which ultimately became Calrose 76 was selected in the M² generation by Dr. Hu in 1971 during his year as a Visiting Scientist at UCD. He also made other selections for short stature and early maturity. After Hu’s return to Taiwan in 1972 Rutger pursued genetic and agronomic evaluation of the mutants (Rutger et al., 1976), resulting in Calrose 76 (Figure 1) (Rutger et al., 1977).

Another cultivar, M-101, was developed by integration of the mutant semidwarf gene into cross-breeding by com- bining it with an early flowering mutant gene, and the gene for glabrous leaves and hulls from the closely related cultivar CS-M3 (Rutger et al., 1979). Keys to the success of this work were 1) inducing mutants in a very good cultivar, Calrose, which needed only a couple of corrections, in this case, semidwarfism and earlier maturity, and 2) immediately evaluating the mutants agronomically and genetically, and 3) then integrating them into conventional crossbreeding efforts.

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Figure 1. The induced mutant Calrose 76, released in 1976, was the first semidwarf table rice cultivar in the USA. It is about 25% shorter than its tall parent, Calrose, and the closely related tall cultivar CS-M3. Calrose 76 and its derivatives have been widely used as the ancestral source of semidwarfism in breeding programs in California, Australia, and Egypt. Calrose 76 carries the sd1 semidwarfing allele.

Concurrently with the cultivar releases Rutger and his students determined that the semi dwarfing gene in Cal- rose 76 was allelic to sd1 from DGWG (Foster and Rut- ger, 1978a), and that it was independent of the widely used gene for glabrous leaves and hulls (Foster and Rut- ger, 1978b). Other studies included inheritance of an early maturity mutant (McKenzie et al., 1978), and inheritance of additional semidwarfing genes (Mackill and Rutger, 1979).

Rice breeding colleagues in California immediately pursued cross-breeding Calrose 76 with the tall cultivar CS- M3 to produce M7 (Carnahan et al., 1978). In cultivar x nitrogen fertilizer rate studies the two semidwarf cultivars Calrose 76 and M7 averaged 14% more grain and 13%

less straw than the tall check cultivar CS-M3 (Figure 2) (Brandon et al., 1981). In farm practice yields of 20-25%

were commonly observed since the higher nitrogen fertil- ity levels resulted in lodging and consequent yield de- creases in the tall cultivars. California growers very quickly began adopting semidwarf cultivars.

By 2005 the total number of California cultivars, including Calrose 76 itself, that traced their semidwarfism an- cestry to Calrose 76 had grown to 13 (Table 1). The most recent of those cultivars, Calamylow-201, not only carries the semidwarf gene but also was itself an induced mutant for a second characteristic, a speciality low amy- lase (ca 6%) type which is expected to be useful for a new developing rice market (McKenzie et al., 2006). So Induced mutations are being pyramided! Calrose 76 an- cestry appears in the pedigrees of 10 additional California cultivars resulting from crosses between the Calrose 76 source and other semidwarf sources, mostly IR8 or DGWG (K.S. McKenzie, Director of California Coopera- tive Rice Research Foundation, personal communication, August 22, 2005). Molecular technology now makes it possible to determine exactly which parent contributed the semidwarf allele gene in such semidwarf x semidwarf crosses. For example, the most successful cultivar in

California for the last two decades, M-202 (Johnson et al., 1986), derived from crossing the Calrose 76 source and the IR8 source, was recently shown to carry sd1 from IR8, while S-101, another cultivar resulting from crossing the two sources, carries sd1 from Calrose 76 (T.H. Tai, Rice Geneticist, Davis, California, personal communication, October 17, 2006).

Figure 2. Averaged over nitrogen fertilizer rates from 60 to 180 lb/acres (67 to 202 kg/ha), the two semidwarf cultivars Calrose 76 and M7 yielded 14% more grain and 13% less straw than the tall cultivar CS-M3 (Brandon et al., 1981).

In Egypt 2 semidwarf cultivars were developed using the Calrose 76 source of semidwarfism (Table 2). In Austra- lia an additional 10 semidwarf cultivars traced their an- cestry to M7, the California glabrous leaf cultivar which received its semidwarfing gene from Calrose 76 (R. Re- inke, Rice Breeder, Yanco Agricultural Institute, personal communication, December 21, 2005).

The experimental semidwarf Short Labelle was selected in California from an M² population grown from bulk M¹ seed of the cultivar Labelle supplied by C.N. Bollich, USDA-ARS, Texas. Short Labelle was determined to be nonallelic to sd1 and was evaluated for productivity in the southern US, but was not released as yields were generally lower than the parent cultivar (McKenzie and Rut- ger, 1986). The semidwarf cultivar Mercury, released in Louisiana, was selected from the cross Short Mars/Nato (McKenzie et al., 1988). Short Mars, a semidwarf selection made at Davis, California, from a mutagenized population of the tall Arkansas cultivar Mars, showed some segregation for maturity and may have resulted from an outcross. Genetic studies indicated that Short Mars car- ried a semidwarfing gene allelic to sd1 (McKenzie et al., 1988).

An interesting spontaneous mutant for elongated uppermost internode, eui, inherited as a recessive tall plant type, was postulated to have breeding value in hybrid rice seed production, that is, tall male plants would be desir- able for pollen dispersal onto short female plants, and

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being recessive, the tall plant type would not be ex- pressed in the desired semidwarf F¹ crop (Rutger and Carnahan, 1981). Allelic eui mutants

were later found to occur in japonica germplasm (Mackill et al., 1994), as well as in indica germplasm (Rutger, 2005).

Table 1. California cultivars for which Calrose 76 served as the ancestral source of semidwarfism

Cultivar Year Pedigree Reference

Calrose 76 1976 Induced mutant of Calrose Rutger et al., 1977

M7 1978 Calrose 76/CS-M3 Carnahan et. al., 1978

M-101 1979 CS-M3/Calrose 76//D31 Rutger et al., 1979

M-301 1980 Calrose 76/CS-M3//M5 Johnson et al., 1980

S-201 1980 Calrose 76/CS-M3//S6 Carnahan et al., 1980

M-302 1981 Calrose 76/CS-M3//M5 Johnson et al., 1981

Calmochi-101 1985 Tatsumimochi//M7/S6 Carnahan et al., 1986

S-101 1988 70-6526//R26/Toyohikari/3/M7/74-Y-89//SD7/73-221 Johnson et al., 1989

M-103 1989 78-D-18347/M-302 Johnson et al., 1990

S-301 1990 SD7/730221/M7P-1/3/M7P-5 Johnson et al., 1991

S-102 1996 Calpearl/Calmochi-101//Calpearl McKenzie et al., 1997

Calhikari-201 1999 Koshihikari/(Koshihikari/S-101)*2 McKenzie, 2001 Calamylow-201 2006 Induced low amylose mutant of Calhikari-201 McKenzie et al., 2006 Table 2. Egyptian rice cultivars for which Calrose 76 served as the ancestral source of semidwarfism

Cultivar Year Pedigree Reference

Giza 176 1989 Calrose 76/Giza172//GZ 242 Badawi, 1999

Sakha 101 1997 Giza 176/Milyang 79 Badawi, 1999

Other rice mutants produced in California included various short stature, early maturing marker gene stocks, and several genetic male steriles (Rutger et al., 1982, 1987;

Mese et al., 1984). An attempt at inducing cytoplasmic male sterility (CMS) in rice with streptomycin was not successful but did result in another genetic male sterile (Hu and Rutger, 1991); a parallel streptomycin study in sunflower resulted in several CMS mutants (Jan and Rut- ger, 1988).

The genetic male steriles were used as a tool to search for apomixis in rice, by interplanting male steriles between rows of world collection rices, producing 3,178 F¹ plants, then looking for abnormal segregation in the resulting F² populations. The male steriles were homozygous for three recessive marker genes, semidwarfism, glabrous leaves, and male sterility. Abnormal segregation of the three genes, specifically for excess of maternal-type plants, would be evidence of apomixis. Although abnormal segregation ratios was observed in 14 out of 3,728 families, detailed analysis indicated these were due to sampling error, and thus not evidence of apomixis (Rut- ger, 1992a).

An environmentally sensitive genetic male sterile mutant recovered from anther culture of Calrose 76 appeared promising for using genetic male sterility in hybrid seed production (Rutger and Schaeffer, 1994). In the long-day environment at Davis, California, this mutant segregated as a recessive male sterile, in the short day winter nursery environment in Kapaa, Hawaii, no segregation occurred,

i.e., genetically sterile plants became fertile. This offered the potential of producing bulk quantities of seeds from

“sterile” plants in the short-day environment, seeds which would produce all-sterile plants in a crossing block in the long-day environment. Although the trait was reproduci- ble in selfed generations, it was not transmitted in con- trolled crosses, thus limiting its utility (Rutger and Schaeffer, 1994).

In the author’s studies on induced mutants, emphasis has been on finding applications, primarily for breeding, so detailed cytogenetic and molecular studies have not been pursued. The rationale behind this decision has been to get the mutants documented and available for others to use, as in the molecular studies that have been reported on sd1 (Monna et al., 2002), eui (Ma et al., 2006) and lpa1 (Andaya and Tai, 2005). Such basic studies have been very helpful in understanding which alleles are being used in breeding (T.H. Tai, Rice Geneticist, Davis, California, personal communication, October 17, 2006).

Meanwhile, the various mutants found over the last 30 years are listed in Table 3.

The California work up to the early 1990s was summarized in IAEA venues (Rutger and Peterson, 1981; Rut- ger, 1984, 1991, 1992b) and elsewhere (Rutger, 1983).

Arkansas tropical japonicas

After the five-year administrative assignment, the author returned to rice research in 1993 as the first Director of the Dale Bumpers National Rice Research Center in

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Stuttgart. Arkansas, and began work on useful applications of induced mutation in southern US rice. Nearly half of the total US rice production is in Arkansas, all with tropical japonica cultivars, primarily long grain rices with intermediate amylose contents (21-23%). At that time no cultivars carrying the semidwarfing gene had been released in Arkansas, although the modern Arkansas cultivars were only 15 to 25 cm taller than semidwarfs from Texas and Louisiana. Therefore considerable effort was directed at inducing and evaluating semidwarf mutants in the tall Arkansas cultivars. Over 100 putative semidwarfs were selected in the next 5 years, but through agronomic evaluation these were reduced to just 12 single recessive gene mutants which equaled their tall parents in yield; those not equaling the tall parent were summarily discarded (Rutger et al., 2004b, 2004c, 2006).

Each mutant was test crossed to the Calrose 76-derived induced sd1 source from California. Surprising, all 12 proved to be nonallelic to sd1 (Figure 3). The fact that none of these 12 mutants gave the 14% or higher yield increase that was customarily observed with sd1 in Cali- fornia, gave further credence to the previously postulated

“sd1 mystique” (Rutger, 1992b).

Figure 3. A semidwarf mutant of LaGrue and its tall parent, LaGrue. As with all 12 semidwarf germplasm mutants that Rutger induced in Arkansas cultivars, the semidwarfing gene in the LaGrue mutant was nonallelic to sd1, the worldwide semidwarfing gene.

In addition to the semidwarf mutants in the tropical japonica germplasm, a recessive early flowering mutant that was about 16 days earlier than its parent was induced in the cultivar LaGrue (Rutger et al., 2004e). Also induced was the low phytic acid mutant lpa1 in the cultivar Kaybonnet (Rutger et al., 2004a). This mutant reduces the phytic acid phosphorus content of rice about 45%, with a concomitant increase in free phosphorus (Larson et al., 2000). Phytic acid phosphorus is largely indigesti- ble for monogastric animals, and phytic acid also inter- feres with iron and calcium uptake, while free phosphor-

rus is digestible. The phytic acid differences are concentrated in the bran portion of the rice grain (Bryant et al., 2005). Thus to get full benefit of the reduction one should eat brown (unmilled) rice, which to date has been a minor use of rice but may increase with current interest in con- suming whole grain cereals. The low phytic acid mutant has about a 10% yield penalty (Rutger et al., 2004a).

Otherwise the mutant is phenotypically identical to its parent, creating seed purity challenges. Therefore the low phytic acid mutant was crossed with an older goldhull- color cultivar, Bluebelle, and low phytic acid, goldhull color recombinants were obtained to form the germplasm designated GLPA, for goldhull low phytic acid (Rutger et al., 2004d). Currently there are no other goldhull cultivars in production in the USA so GLPA has identity preserva- tion in the field, in the farm truck and in the grain eleva- tor.

Two dominant genetic male sterile mutants were induced, one in the long grain cultivar Kaybonnet and one in the medium grain cultivar Orion (Zhu and Rutger, 1999).

Genetic male sterility is usually advocated for population improvement schemes such as recurrent selection. The principal merit of dominant genetic male sterility is that the sterility recurs every generation, in contrast to recessive male sterility, where the male sterility recurs in every second generation.

Another example of integration of mutants was provided by development of aromatic se germplasm as a semidwarf (s), early maturing (e) recombinant from a cross between a late maturing semidwarf mutant, DM 107-4, and the early maturing tall cultivar Kashmir Basmati (Rutger and Bryant, 2004). Both of the parents were induced mutants of Basmati 370 developed in Pakistan (Awan, 1984). The aromatic se germplasm retains the aroma and cooking quality of the original basmati source. Yield of aromatic se has been low relative to its tall parent in Ar- kansas (Rutger and Bryant, 2004). Awan and Cheema (1999) reported that DM 107-4 has a semidwarfing gene nonallelic to sd1. Therefore attention in Arkansas has shifted to recombinants from crossing another late maturing semidwarf mutant, DM2 (Awan, 1984), with Kashmir Basmati, in hopes that DM2 may carry the “mystical”

sd1. Evaluation of F⁴ generation early maturing, semidwarf recombinants show this new germplasm, designated aromatic se2, indeed has higher yield potential than the first germplasm, aromatic se (Rutger, unpublished).

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Table 3. List of rice mutants collected in California and Arkansas California mutants

semidwarf Calrose CI 9966 Calrose 76 sd1 Rutger et al., 1977

“ “ CI 11033 D66 sd2 Foster and Rutger, 1978a; Rutger et al, 1979 “ “ CI 11034 D24 sd4 Mackill and Rutger, 1979; Rutger et al., 1979 “ “ D32 sd1, D23sd4, D25sd4 Mackill and Rutger, 1979

early flowering Calrose CI 11037 D18 McKenzie et al., 1978; Rutger et al, 1979 “ “ “ CI 11038 D31 Rutger et al, 1979

doubledwarf Calrose CI 11036 DD1 sd1 +sd2 “

semidwarf Colusa CI 11035 sd unknown “

short Labelle sd? ≠ sd1* McKenzie et al., 1986

short stature M5 CI 11045 sd? ≠ sd1 Rutger et al., 1982

short stature M5 CI 11046 sd? ≠ sd1 “

short stature Maxwell CI 11047 sd? ≠ sd1 “

short stature WC 1403 CI 11048 sd unknown “

narrow leaf semidwarf CI 11049 sd? ≠ sd1 “

short stature Tsuru Mai CI 11050 sd unknown “

early flowering Calrose 76 CI 11051 sd1 “

early flowering M5 CI 11052 ef unknown “

early flowering S6 CI 11053 ef unknown “

early flowering Terso CI 11054 ef unknown “

Calady, Earlirose, Caloro,CS-M3

male steriles 4 recessive male steriles Trees and Rutger, 1978

elongated uppermost internode CI 11055 eui Rutger and Carnahan, 1981 early flowering S-201 PI 506219 ef unknown Rutger et al., 1987

light green panicle M-101 PI 506221 lgp “

yellow-green panicle ESD7-3 PI 506222 yp “

waxy M-101 PI 506223 wx “

goldhull M101 PI 506224 gh “

streptomycin-induced male sterile PI 543853 ms Hu and Rutger, 1991 Arkansas mutants

dominant male sterile KBNT

1789 GSOR 1 Ms Zhu and Rutger, 1999

dominant male sterile Orion 1783 GSOR 2 Ms “ “ recessive male sterile Cypress

1819 GSOR 3 ms “ “

semidwarf KBNT 4 PI 632276 sd? ≠ sd1 Rutger et al., 2004b

semidwarf KBNT 5 PI 632277 sd? ≠ sd1 “

semidwarf LGRU 12 PI 632278 sd? ≠ sd1 “

semidwarf ADAR 10 PI 632280 sd? ≠ sd1 “

semidwarf ORIN 172 PI 632281 sd? ≠ sd1 “

semidwarf ADAR 22 PI 632951 sd? ≠ sd1 Rutger et al., 2004c

semidwarf KATY 1 PI 632952 sd? ≠ sd1 “

semidwarf KBNT 11 PI 632953 sd? ≠ sd1 “

semidwarf DR1 PI 642749 sd? ≠ sd1 Rutger et al., 2006

low phytic acid KBNT lpa1 PI 632282 lpa1-1 Rutger et al., 2004a goldhull low phytic acid PI 632954 lpa1-1 + gh Rutger et al., 2004d

Guichao 2 eui GSOR 11,

PI 634574 eui Rutger, 2005

early flowering LaGrue GSOR 7,

PI 632957 ef unknown Rutger et al., 2004e Arkansas indicas

By the mid-1990s it had become evident that indica germplasm had greater yield potential in the southern US than tropical japonica germplasm (Eizenga et al., 2006), although indicas generally had higher amylose contents than the intermediate amylose (21-23%) level desired for US markets. Therefore the first approach was to cross a

very early indica from China, Zhe 733, which had high amylose, with intermediate amylose indicas from IRRI that were about a month too late in maturity for Arkansas (Figure 4). The IRRI lines, which very closely approach US long grain quality standards, were graciously supplied by G. S. Khush of IRRI (G. S. Khush, personal communication, December 20, 1995). Nine early maturing, intermediate amylose recombinants, indica-1 to indica-9,

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from this program were released (Rutger et al., 2005), but these still suffered from lower whole grain milling yield (white rice) than US tropical japonicas. Meanwhile it had become apparent that the IRRI germplasm lines used in these crosses had both the appropriate intermediate amylose levels and high whole-grain milling yields. It was then hypothesized that induction of earlier maturity in the IRRI materials could enhance their value for the US. This indeed proved to be the case, resulting in induction and evaluation of four early flowering mutants, indica-10 to indica-13, which were 18 to 28 days earlier than their parent (Figure 5). The mutants were nearly as early as local tropical japonica cultivars, and most importantly, the mutants retained not only the desired intermediate amylose levels of the parents but also high whole-grain milling yields, giving indica germplasm that is competitive in quality with US long grains (Rutger et al., 2007) (Figure 6).

Figure 4. J. Neil Rutger in his “laboratory” in September 2002, at Stuttgart, Arkansas, observing an early flowering indica germplasm line (left) and its late flowering parent (right).

Meanwhile, the induced low phytic mutant, KBNT lpa1- 1, was crossed with Zhe733 to determine the chromo- some location of the lpa1-1 gene (Larson et al., 2000).

Subsequently this japonica/indica population was advanced to the F¹⁰ generation in order to create a mapping population (Rutger and Tai, 2005). This mapping population has been distributed to interested colleagues for study of blast disease and insect resistance.

A series of 21 early flowering mutants was induced in the famous blast resistant cultivar from Colombia, Oryzica llanos 5 (Roca et al., 1996), another indica cultivar that is a month too late for the US. All 21 mutants, which were from 24 to 40 days earlier than the parent cultivar, retained the parental resistance to six blast isolates; the group has been narrowed down to two for germplasm release (Rutger and Lee, 2007).

Figure 5. The induced early flowering mutant indica-12 (right) is 28 days earlier than its IRRI parent IR53936-60-3-2-3-1 (left), making it useful for the US since the IRRI parent is about a month too late when grown in the US.

Figure 6. The four induced early flowering indica mutants, indica-10, indica-11, indica-12 and indica-13 have head rice (whole kernel) yields similar to the japonica check cultivar Francis. This is the first time that indicas with high head rice yield.

Genetic stocks – Oryza collection (GSOR)

Development of the various mutants in the US led to the 2003 establishment of the Genetic Stocks-Oryza Collec- tion (GSOR) at the DB NRRC in Stuttgart, Arkansas, USA. The GSOR is a much-needed effort since introduc- tions of rice germplasm from overseas is hindered by strict quarantine introduction procedures within our country, which are directed at keeping unwanted diseases and pests out. Therefore we set out to make our own collection of genetic stocks. The first rice genetic stocks contributed to the collection were GSOR 1, 2, and 3, which were two dominant and one recessive genetic male sterile mutants induced at the DB NRRC (Zhu and Rutger, 1999). Sixteen more previously developed entries from California and Arkansas, including induced mutants for genetic male sterility, early flowering, semidwarfism, and elongated uppermost internode were included in the ini-

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tial contributions to the GSOR (Rutger and Carnahan, 1981; Rutger et al., 1982, 1987; McKenzie and Rutger, 1986; Rutger, 2005). Currently the GSOR has 902 entries including: a lesion mimic mutant, GSOR 20 (Jia, 2005), the Stuttgart-developed japonica/indica mapping population with 355 lines (Rutger and Tai, 2005), a second mapping population with 325 doubled haploid lines from Louisiana State University (Chu et al., 2006), and the former “Jodon collection” (Jodon, 1977). A set of 191 Hokkaido University mutants, donated by Japan’s Dr.

Toshiro Kinoshita, via Dr. Susan McCouch, Cornell Uni- versity (T. Kinoshita, personal communication, Novem- ber 22, 2005), has been entered and is part of the 902 total. Four indica genetic stocks, apoptosis, chives, extreme dwarf, and gold leaf, designated as GSOR entries 21, 22, 23, and 24, respectively, were added in 2006 (Rutger and Bernhardt, 2006), and more DB NRRC-developed genetic stocks are in the pipeline. A very recent example mutant is a giant embryo mutant, in the long grain cultivar Drew (Figure 7). This recessive mutant increases oil content from the 2.7% level in the parent cultivar, to 3.7% in the mutant. The mutant, preserved as GSOR 25, may have potential for brown rice consumption, assum- ing that the increased oil content will have a favorable effect on taste (GSOR, 2006). Two indica doubledwarf mutants also are recent GSOR additions. These are of interest because in the work with indicas, all of which apparently carry the DGWG-TN1-IR8 source of sd1, it was usually observed these semidwarfs were 10 cm or so taller than tall Arkansas check cultivars (Rutger et al., 2005, 2007). Since the indicas generally are more suscep- tibleto lodging than local checks, short plants were sought in mutagenized populations of earlier releases.

Figure 7.The giant embryo mutant (left) has an embryo 44 % larger than the long grain parent cultivar Drew (right), and has whole kernel oil content of 3.7% compared to 2.7% for the parent. This mutant, which has been placed in the Genetic Stocks—Oryza Collection as GSOR 25, may have value for consumption as whole grain brown rice, which is a growing market in the US.

Thus one was found in a sister line of indica-9, and another one in indica-12. These “doubledwarf” mutants, which are 15 to 20 cm shorter than their respective sin-

gle-dwarf parents, were placed in the GSOR collection, as GSOR 27 and 28, respectively (GSOR, 2006). Finally, an indica mutant population, GSOR 26, which segregates for albinos, was preserved to serve as an elementary school teaching project, that is, 3 normal: 1 albino segregation can be observed by germinating the seeds for 5 to 7 days (GSOR, 2006). Entries in the GSOR may be

viewed at the GSOR homepage

http://ars.usda.gov/Main/docs.htm?docid=8318.

Cooperation with IAEA

The author has been fortunate to have numerous opportu- nities to cooperate with IAEA over the past 30 years, through participation in international meetings and /or workshops, beginning in 1977:

1. October 7-9, 1977. Presented invitational paper on

“Utilization of induced mutants for short stature and early maturity in japonica rice” at the FAO/IAEA Regional Seminar on Improvement of Rice Production Through Research Using Nuclear Techniques, Jakarta, Indonesia.

2. March 1-14, 1981. Participated in FAO/ the IAEA RCM on Evaluation of Mutant Stocks for Semid- warf Plant Type as Cross-Breeding Materials in Cereals, and in the FAO/IAEA International Sym- posium on Induced Mutations as a Tool for Crop Improvement. Vienna, Austria.

3. September 15, 1983. Hosted FAO/ IAEA RCM on Evaluation of Mutant Stocks for Semidwarf Plant Type as Cross-Breeding Materials in Cereals.

Davis, California.

4. December 13-21, 1985. Participated in Fourth RCM on Evaluation of Semidwarf Mutants for Cross-Breeding. Rome, Italy.

5. June 16-23, 1990. Presented invited paper on “Mu- tation breeding of rice in California and the USA, at the FAO/IAEA International Symposium on the Contribution of Plant Mutation Breeding to Crop Improvement. Vienna, Austria.

6. May 6-14, 1994. Participated in the First FAO/IAEA RCM on Radiation-induced Mutations and other Advanced Technology for the Production of Seed Crop Mutants Suitable for Environmen- tally Sustainable Agriculture. Vienna, Austria.

7. June 19-23, 1995. Participated as invited advocatus diaboli at the FAO/IAEA Symposium on the Use of Induced Mutations and Molecular Techniques for Crop Improvement. Vienna, Austria.

8. November 6-10, 1995. Participated in the Second FAO/IAEA RCM on Radiation-induced Mutations and other Advanced Technology for the Production of Seed Crop Mutants Suitable for Environmen- tally Sustainable Agriculture. San Jose, Costa Rica.

A major effort in the author’s induced mutation career was as a consultant in the IAEA program for Evaluation of Cereal Crop Mutants (ARCAL

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XXIA) from 1995 to 2001 in six Latin American countries, organized by M. Maluszynski, former Head of the Plant Breeding Section at IAEA.

Twenty three rice mutants, including one from the US, were evaluated in multi-location yield trials by cooperators in Brazil, Colombia, Costa Rica, Cuba, Guatemala, and Uruguay (Blanco et al., 2000). In general, the 22 indica mutants performed better in these tropical and subtropical environ- ments than did the one US mutant, OR172, which was in a japonica background. The Latin Ameri- can model subsequently was adopted for similar mutant evaluations organized by Maluszynski in 11 Asian countries (Eizenga et al., 2004). Both the Latin American and Asian evaluation trials showed the usefulness of mutant germplasm in contributing to food security in participating countries (Blanco et al., 2000; Eizenga et al., 2004). In- dividual country meetings are summarized in items 9 to 14, below.

9. December 2-9, 1995. Began service as a coordinator for evaluation of 23 rice mutants, 10 parent lines, and a local check, by scientists in Brazil, Co- lombia, Costa Rica, Cuba, Guatemala, and Uru- guay. I contributed one of my tropical japonica semidwarfs to this otherwise all-indica collection of mutants. Campinas, Brazil.

10. October 19-26. 1996. Continued as rice ex- pert/coordinator. Buenos Aires, Argentina.

11. July 5-12, 1997. Continued as rice ex- pert/coordinator. San Jose, Costa Rica.

12. March 22-29, 1998. Continued as rice ex- pert/coordinator. Pointe del Este, Uruguay.

13. November 7-12, 1999. Continued as rice ex- pert/coordinator. Guatemala City, Guatemala.

14. December 11-15, 2000. Continued as rice ex- pert/coordinator. Guatemala City, Guatemala.

15. August 24-28, 1998. Hosted FAO/IAEA RCM on Radiation-induced Mutations and other Advanced Technology for the Production of Seed Crop Mu- tants Suitable for Environmentally Sustainable Ag- riculture. Stuttgart, Arkansas, USA,

16. October 4-8, 1999. Participated in First FAO/IAEA RCM on “Molecular characterization of mutated genes controlling important traits for seed crop improvement.” Vienna, Austria.

17. June 3-10, 2000. Invited participant in IAEA Re- gional Training Course on New Frontiers of De- veloping and Handling mutants. Hangzhou, China.

18. June 10-14, 2002. Participated in FAO/IAEA RCM on “Molecular characterization of mutated genes controlling important traits for seed crop improvement.” Krakow, Poland.

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Research Article

Development of Three Groundnut Varieties with Improved Quantitative and Qualitative Traits through Induced Mutation

M.A. Hamid¹, M.A.K. Azad² and M.A.R. Howlider³

1Bangladesh Institute of Nuclear Agriculture, P.O. Box 4, Mymensingh 2200, Bangladesh

2Plant Breeding Division, Bangladesh Institute of Nuclear Agriculture, P.O. Box 4, Mymensingh 2200, Bangladesh

3Plant Pathology Division, Bangladesh Institute of Nuclear Agriculture, P.O. Box 4, Mymensingh 2200, Bangladesh Abstract

With a view to develop high yielding, bold seeded and disease resistant varieties of groundnut, 200 dry seeds of an established mutant, Mut-6, was irradiated with 200 Gy gamma rays. All the M1 plants were harvested and kept separately. In M2-M4

generations, selection and evaluation were made following high mean and high/low variances compared to the check cultivar Dhaka-1 (the parent of Mut-6). In M4 generation, 16 true breeding lines were obtained. Through preliminary, advance, zonal and farmers’ field trials during 1997-1999, three mutant lines M6/20/42-M(2), M6/20/44-3 and M6/20/62-4, were identified to be dwarf, high yielding, bold podded and seeded and moderately resistant to cercospora leafspot, rust and collar rot diseases, compared to the check variety, Dhaka-1. Moreover, these mutants have higher oil contents and higher/similar protein contents. The National Seed Board has registered these mutant families in 2000 as BINAchinabadam-1, BINAchina- badam-2 and BINAchinabadam-3, respectively, for commer- cial cultivation by the farmers.

Key words: groundnut; induced mutations; new cultivar Introduction

Groundnut (Arachis hypogaea L.) is an important oil and food crop, currently being grown on approximately 17 million hectares of land worldwide with the production of 23.2 million metric tons [1]. Globally, it is the third major oil seed crop next to soybean and cotton. India, China and the United States of America have been the leading producers for over 25 years and produce about 70% of the total production. In Bangladesh, it ranks third after rapeseed-mustard and sesame based on both acreage and production despite per hectare yield is the highest in groundnut (1150 kg) [2]. It is a multipurpose crop and can help reduce edible oil, food and fodder shortages of the country. Apart from its rich oil content (45 to 50%), groundnut seeds are good source of proteins (25 to 30%), carbohydrates (20%) and vitamins E and B. A pound of peanuts provides food energy that is equivalent to 2 pounds of beef, 9 pints of milk, or 36 medium sized eggs [3]. For its high digestibility it is an excellent component of children’s food. Being a legume it fixes atmospheric nitrogen to soil through its nodule bacteria and thus keeps environment most friendly [4]. Groundnut yield is one of the lowest in Bangladesh compared to the very high yields in the developed countries, particularly in the USA (2400-3200 Kgha^-1). The low genetic potential, smaller pod size and high susceptibility to disease and insect- pests of the widely grown land race, Dhaka-1, mostly attribute this low yield. Mutation breeding technique is one of the important accessories of the main stream plant breeding. Compared to conventional methods, it saves nearly half the time to create a new cultivar. Pleiotropic

effects are very common and help fix true breeding lines even in M³/M⁴ generations. Genetic improvement of any yield attributes, both quantitative and qualitative in nature, has been successful through this technique [5-9].

Keeping these in mind this study was initiated to develop high yielding, bold seeded and disease resistant varieties using mutation breeding technique.

Materials and methods

Two hundred dry seeds of an established mutant, Mut-6, were irradiated with 200 Gy dose of gamma rays. The mutant, Mut-6, was originally developed during 1980- 1986 by treating seeds of Dhaka-1, a widely cultivated cultivar, with gamma rays at a dose of 400 Gy. Treated seeds of Mut-6 were immediately sown for M¹ population development and at maturity plants were harvested separately and dry pods were kept for growing M² population. The following year, M²-plant-progeny-rows were grown with check-rows of Dhaka-1 in every 15-row in- terval. In this generation, the most competitive ten plants from each and every M²-plant-progenies including Dhaka-1 check were recorded. Approximately 200 families that showed high mean yields with either high or low variances compared to Dhaka-1 were selected. Exactly similar procedure of selection was practiced in M³ generation and 98 out of 200 families were selected on the basis of high means and low variance compared to the mean yield and variance of Dhaka-1. An observational yield trial was then conducted in M⁴generation during the winter season of 1997 with these 98 mutant families. This trial identified 16 true breeding lines with higher mean yields.

Preliminary yield trial (M6 generation)

These 16 true breeding families, along with their grand parent, Dhaka-1, were put into preliminary yield trial in Kharif-II season of 1997 at Mymensingh. The experiment followed a randomized complete block design with three replications having plot sizes of 4.5m x 1.0m. Seeds were sown at 15cm distances within rows of 45 cm apart. All plots were fertilized with 40kg N, 120 kg P²O⁵ and 120 kg K²O ha^-1during final land preparation. Recommended cultural practices were followed as and when necessi- tated. Data on different yield attributes were recorded only from 10 randomly competitive selected plants.

Moreover, yield from 1.4 m² area was recorded. Disease reaction data on cercospora leaf spot (Cercospora ara- chidicola), rust (Puccinia arachidis) and collar rot (As- pergillus niger) were also recorded from this experiment.

Fifteen randomly selected plants from each plot were scored before 15-20 days of harvest for cercospora leaf

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spot and rust as per standard 9 point rating scale developed by Subrahmanyan et al. [9]. For collar rot, data on germination, pre-and post emergence seedling deaths were recorded and finally, graded following standard

scale (1-9) developed by Nene et al. [10] and presented in Table 1.

Table 1. Means pod yield over three locations and different yield attributes over two locations in some elite groundnut mutants

Mutant PH

(cm) PBPP (no.) NPP

(no.) HPW

(g) HKW

(g) Shelling

(%) Pod yield (kg ha^-1)

Mean Mymensingh Ishurdi Natore

M6/20/42-M(2) 31.8 5.0 24.2 85.7 47.6 73.4 2594 3734 2070 1976

M6/20/44-3 27.5 5.0 25.1 83.4 40.1 68.6 2486 3168 2423 1866

M6/20/62-4 27.6 5.2 22.3 86.0 42.3 68.9 2371 2991 2184 1939

Dhaka-1(c) 37.8 4.8 26.4 67.6 34.1 70.0 1922 2610 1949 1207

Zhingabadam (c) 49.5 3.7 21.9 75.3 29.0 65.6 1734 2448 1383 1372

LSD(0.05) 04.6 0.5 5.0 9.6 2.8 3.9 197 478 373 179

PH=plant height; PBPP=number of primary branches per plant; HPW/HKW=100 pod/kernel weight

Advanced yield trial (M6 generation)

An Advanced Yield Trial was conducted with 7 M⁶mutant families along with two check cultivars, Dhaka-1 and Zhangabadam, during the winter season of 1998 at BINA farm, Mymensingh. The experiment followed a randomized complete block design with three replications. Seeds were sown in unit plot sizes of 4.5 m x 1.35m at 15 cm distances within rows of 45 cm apart. All plots were fertilized following BARC Fertilizer Recommendation Guide (1995) during final land preparation. The experiment followed rainfed condition and recommended cultural practices. Pod yield data was taken from an area of 1.42 m²/plot having uniform population size of 21 plants.

Farmers’ field trial

Farmer’s field trials were conducted with 3 elite M⁷ mutant families together with two check varieties, during winter season of 1998-99, at Mymensingh, Ishurdi and Natore. The experiment followed randomized complete block designs with three replications, in each location.

Seeds were sown in unit plot sizes of 4.05m x 2.25m at 15 cm plant distances within rows of 45 cm apart.

Fertilizers were applied following BARC Fertilizer Rec- ommendation Guide (1995) during final land preparation.

The crop was raised in rainfed condition where recommended cultural practices were followed. Pod yields were gathered from an area of 3.375 m²/plot. Reaction to cercospora leaf spot, rust and collar rot diseases were recorded following the procedure as described in preliminary yield trial. Oil and protein contents were also determined [11, 12]. The yield in various tests was trans- formed into Kg ha^-1 and was subjected to proper statisti- cal analyses by following the given design.

Results and discussion

Performance in preliminary yield trial

The check cultivar, Dhaka-1, had the tallest height and was significantly different from some of the mutant lines,

e.g., M6/20/60-1, which was only less than half of Dhaka-1 (Table 1). Interestingly, we found most of the mutant lines were higher than Mut-6, although not always significant (Table 2). Variations of other agronomic traits and yield attributes and disease resistance were also observed and seven lines were selected for advanced yield trial.

Performance in advanced yield trial

Zhingabadam had the largest pod size while Dhaka-1 had the least sized pod. The mutant family M6/20/62-4 had shown significantly higher pod size than Dhaka-1 con- trol. Shelling percentage had not differed significantly among the mutant families and check cultivars. Mutant genotype M⁶/20/62-4 had produced the highest yield and did not differ from the 5 other mutants and two check cultivars. Results of this advance trial were further as- sessed through pool analyses with yield and yield attribute records of the previous preliminary trial for proper evaluation of the mutant families for future farmer’s field trial.

It could be seen clearly that not all mutant lines had con- sistently high yield in preliminary and advanced yield trial (Table 1, 2). Based on comprehensive consideration of earliness, mature pod numbers, dwarf types, shelling percentages and yields, three lines M⁶/20/42-M(2), M⁶/20/62-4, M⁶/20/44-3 were selected.

Performance in farmer’s field trials

The yields of the three mutants, M⁶/20/42-M(2), M⁶/20/44-3 and M⁶/20/62-4, were significantly higher than the two check varieties, Dhaka-1 and Zhingabadam, both on average and in each location (Table 5).

M6/20/42-M(2), for example, out-yielded on average the two check cultivars Dhaka-1 and Zhingabadam by 35.0%

and 49.6%, respectively. All mutants and check cultivars showed resistance (R) to moderately resistance (MR) to all the three most prevailing diseases, i.e. collar rot, CLS and rust.

Plant Mutation Reports Vol. 1, No. 2, December 2006 | IAEA

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